Artifactual sulfation of silver-stained proteins: implications for the assignment of phosphorylation and sulfation sites.
Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.
Gharib
Marlene
M
Institute of Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada.
Marcantonio
Maria
M
Lehmann
Sylvia G
SG
Courcelles
Mathieu
M
Meloche
Sylvain
S
Verreault
Alain
A
Thibault
Pierre
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D016477:Artifacts; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010751:Phosphopyruvate Hydratase / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016622:Silver Staining / Q000379:methods*; D013431:Sulfates / Q000378:metabolism*; D013885:Thiosulfates / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D010455:Peptides; D011506:Proteins; D013431:Sulfates; D013885:Thiosulfates; D048052:Mitogen-Activated Protein Kinase 3; D010751:Phosphopyruvate Hydratase; C017717:sodium thiosulfate